DBA/2J and C57BL/6J mice have been shown previously to differ largely in their susceptibility to H1N1 (PR8M) influenza A virus [9
]. Here, we expanded these studies and utilized the BXD recombinant inbred set of mouse strains to map the genomic regions that are responsible for differences in these two mouse strains. We monitored three phenotypic traits, body weight over time, survival over time and mean time to death to identify quantitative trait for influenza resistance. Two significant QTLs, Qivr5
, were found on chromosomes 5 and 19, respectively. Furthermore several suggestive QTLs, Qivr2
were observed in at least two traits and at two days on chromosomes 2, 16 and 17, respectively. Composite mapping revealed an additional almost significant QTL at distal chromosome 16, Qivr16
A similar analysis for host resistance to influenza has been performed previously after infecting 66 BXD strains with H5N1 influenza virus. This study reported three significant QTLs on chromosomes 2, 7, and 17 [12
]. Thus, none of these significant QTLs overlaps with the QTLs identified in our analysis. Five of the strains that were resistant (all infected mice survived) in our study were also resistant in the study of [12
] where a total of 14 strains were found to be resistant. Five strains that were highly susceptible in our study (100% of infected mice died) were also highly susceptible in the study by [12
] of a total of 26 susceptible strains. Furthermore, five strains that were resistant in our study were susceptible in the study by [12
]. Thus, there is also not much overlap between the two studies with respect of susceptible and resistant strains. The differences between the two studies are most likely explained by the use of two different influenza virus subtypes. The H1N1 virus from our study represents a subtype with a monobasic hemagglutinin (HA) cleavage site, whereas the H5N1 which was used in the study by Boon et al. is a subtype with a polybasic HA cleavage site. The cellular tropism of these two subtypes for virus replication and processing is quite different, because monobasic viruses are dependent on cell-specific proteases for the processing of the HA whereas polybasic subtypes can be processed by more ubiquitously expressed host proteases, e.g.
]. Therefore, the contribution of host factors to susceptibility may be different between H1 and H5 containing virus subtypes.
Another study described the genetic mapping of susceptibility and resistance factors after infecting a panel of 29 AxB / BxA congenic strains with a mouse-adapted H3N2 influenza virus [33
]. The AxB / BxA congenic strains were generated from a cross of susceptible A/J and resistant C57BL/6J parental mouse strains. The authors found three major QTLs on chromosomes 2, 6 and 17. The QTL on chromosome 17 overlaps with the Qivr17-2
locus which we found in our study. Furthermore, the candidate gene Pla2g7
that was identified in their study was also detected as candidate gene in our analysis (see below).
The influence of genetic factors determining the host response to H1N1 influenza virus infections was also examined in mice of the pre-Collaborative Cross collection [34
]. In this study, gene expression levels in extreme responders were used to identify expression QTLs (eQTL). One gene that exhibited a cis-eQTL, Sik1
(salt inducible kinase 1), was located in the Qivr17-2
interval from our study, and we also identified it as potential quantitative trait gene (Table
). This gene is associated with the GO terms ‘negative regulation of transcription from RNA polymerase II promoter, regulation of cell differentiation, and protein kinase cascade’. However, no specific infection-related functions have been yet described for this gene.
One of the most interesting findings in our study was the time-dependent effect of QTLs which we observed in the body weight and survival traits. The peak QTLs for the two significant QTLs, Qivr5 and Qivr19, were found at different times p.i., day 6 and day 8, respectively. In addition, the effects of both QTLs were not only evident at the times p.i. where they exerted the significant peak QTL signals but also several days before and after the peak. Furthermore, for the suggestive QTLs, also time-dependent effects were observed. These results suggest that the causal genes underlying different QTLs act at different time points of the host defense.
Most interestingly, Qivr5 as well as Qivr19 represent a positive influence on body weight, survival and MTTD from the DBA/2J haplotype, the susceptible strain. These findings indicate that genomic regions from the susceptible parent are able to increase resistance when combined with the resistant parental genome. We are now analyzing several BXD strains that were more resistant than the parental C57BL/6J mice in more detail. One possible mechanism to explain such an effect may be that an activator (secreted ligand or transcription factor) is expressed in susceptible DBA/2J mice but the corresponding target (receptor or regulated gene) is mutated. On the other hand, in C57BL/6J mice, the target but not the activator may be mutated. If the wild type alleles are now coming together in a BXD strain, the functional activator finds its functional target and thereby an increased resistance state is achieved.
Both composite and interaction mapping revealed many genetic interactions between C57BL/6J and DBA/2
J alleles. Thus, many genomic regions from the parental strains are able to contribute to the host response and this effect depends strongly on the allele combinations in the respective QTLs. These observations may be studied further in double congenic mouse lines.
We subsequently analyzed the five QTL intervals, Qivr2
in more detail to identify genes that may be causal for resistance or susceptibility. In total, 830 genes are located in these intervals. We narrowed down the total number of genes to 31 candidates (Table
) by using additional information, such as temporal expression after PRM8 infection (Pommerenke et al., PLoS ONE, in press), cis-eQTLs in non-infected lungs [26
], differences in expression between DBA/2J and C57BL/6J [23
], and sequence variants in the coding regions.
contains the candidate gene Eif2ak1
(eukaryotic translation initiation factor 2 alpha kinase 1) that is a member of eIF2alpha kinases which have been associated with anti-viral host responses [35
]. Boon et al. described another eIF2alpha kinase, Eif2ak2
(eukaryotic translation initiation factor 2-alpha kinase 2), in the Qirv17
locus after infection with influenza H5N1 [12
plays a critical role in modulating immunoglobulin expression during RSV infection. In addition Eif2ak2
knock-out mouse mutants are more susceptible to influenza infections [36
]. We have initiated the generation of a congenic mouse lines for the chromosome 5 interval to verify and further characterize the effect of this region for resistance to influenza infection.
contains two candidate genes, Itgb6
(integrin beta 6) and Ifih1
(interferon induced with helicase C domain 1), with known functions in the host defense to viral infections. Itgb6
mouse knock-out mutants exhibit severe pneumonia and an increase in granulocyte recruitment to the lung [38
]. The protease-activated receptor 1-mediated enhancement of Itgb6-
dependent TGF-beta activation has been proposed to represent one mechanism by which activation of the coagulation cascade contributes to the development of acute lung injury [39
]. The Ifih1
gene is also known as MDA5 (Melanoma Differentiation-Associated protein 5). IFIH1 is part of the RIG-I-like receptor (RLR) family, which function as pattern recognition receptors and are activated upon binding of virus dsRNA [40
]. IFIH1 functions as cytosolic receptor that leads to the selective activation of type I IFN genes and is indispensable for sustained expression of IFN in response to paramyxovirus infection [41
mutant knock-out mice exhibit an impaired response to different viral pathogens [43
contains two potential genes with known functions in the host defense and lung function, Robo1
(roundabout homolog 1 (Drosophila)) and Nrip1
(nuclear receptor interacting protein 1). DBA/2J mice carry a frame shift mutation in the Robo1
gene which might lead to an impaired function of the encoded protein. Robo1
has been described to be involved in guidance and migration of axons, myoblasts, and leukocytes in vertebrates (e.g.
but is also expressed in the developing lung [48
knock-out mutants exhibit a delayed lung maturation and bronchial hyperplasia. The latter results suggest that Robo1
may be involved in maintaining proper lung function and it may become essential when lung epithelium is destroyed during an influenza infection. Nrip1/Rip140
functions as a co-activator for cytokine gene promoter activity via direct protein-protein interactions with the NFkappaB subunit RelA and histone acetylase cAMP-responsive element binding protein (CREB)-binding protein (CBP) [49
]. It is involved in modulating pro-inflammatory responses in macrophages [50
represents a positive influence of the C57BL/6
J genotype on body weight, survival and MTTD. This QTL is located in a gene-rich region which carries many genes that are involved in the host immune response, in particular the H2
histocompatibility genes which are involved in antigen presentation [51
]. Therefore, many candidate genes are found in the Qivr17-2
region. The Lst1
(leukocyte specific transcript 1) gene is of special interest because the DBA/2J allele mice carries a single nucleotide deletion in the first exon resulting in a frame shift of the open reading frame. This mutation most likely results in a non-functional Lst1 protein in DBA/2
J mice. We confirmed the presence of the deletion by sequencing the parental DBA/2J and some BXD strains carrying the DBA/2J allele. The wild type allele was confirmed in C57BL/6J mice and in some BXD strains carrying the C57BL/6J allele. In humans, LST1
plays a role in the regulation of the immune response to inflammatory diseases such as rheumatoid arthritis, microbial infection or Rubella vaccine-induced immunity [52
]. Also, Lst1
is up-regulated after influenza A infection in C57BL/6J mice starting at day 2 and exhibits a strong peak of expression at day 8 p.i. Pommerenke et al., 2012 (Pommerenke, C., E. Wilk, B. Srivastava, A. Schulze, N. Novoselova, R. Geffers, and K. Schughart. 2012. Global transcriptome analysis in influenza-infected mouse lungs reveals the kinetics of innate and adaptive host immune responses. PLoS ONE. 7:e41169.). Thus, the expression profile and known functions of Lst1
fit well with a possible critical role for the host defense to influenza A virus. We initiated the generation of knock-out mice to evaluate the role of Lst1
in more detail. In addition, a second, most interesting candidate, Pla2g7
(phospholipase A2, group VII (platelet-activating factor acetylhydrolase, plasma)) was identified in the Qivr17-2
interval. In humans, increased activities of certain variants of PLA2G7 were associated with early coronary atherosclerosis and with endothelial dysfunction, but the gene may also exert an anti-inflammatory function [56
]. The Pla2g7
gene was also identified as a potential candidate gene for susceptibility against infections with H3N2 influenza virus [33
expression levels in susceptible A/J mice were higher than in resistant C57BL/6J mice after infection with H3N2 virus [33
]. We also showed previously that Pla2g7
exhibits a cis-eQTL between C57BL/6J and DBA/2J in non-infected lungs where the DBA/2J allele shows high levels of expression [26
which was identified by [33
] as potential candidate of Qivr17-2
also exhibits a cis-eQTL in non-infected BXD mice [26
] but was not found to be regulated in C57BL/6 mice after infection (data not published). Tapbp
(TAP binding protein) plays a major role in the antigen processing and MHC class I presentation by stabilizing the TAP peptide transporter, e.g.
]. Also, Tap2
(transporter 2, ATP-binding cassette, sub-family B (MDR/TAP)) gene is involved in antigen processing and presentation [63
(general transcription factor II H, polypeptide 4) encodes a general transcription factor. Recruitment and activation of Gtf2h4
represents a rate-limiting step for the emergence of HIV from latency and sequence variants have been associated with multiple sclerosis [67
Within the Qivr19
interval, only one gene, Hps1
(Hermansky-Pudlak syndrome 1 homolog (human)), has been associated with the host responses to infection. Mice carrying a natural mutation in the Hps1
gene showed an increased inflammatory response in alveolar macrophages after intranasal challenge with LPS [70
The GeneNetwork database allows searching for other phenotypic traits that exhibit a genome-wide significant (LRS
18) within the Qivr
intervals identified by our study. Two phenotypic traits, related to neuronal responses (trait ID 11285) and body weight changes (trait ID 12838), are located to the Qivr16
locus. Also, the Qivr17-2 interval contained significant QTLs for other traits. Two traits are related to host infectious diseases, ‘Ectromelia virus survival’ (ID 12672) and ‘Chlamydia psittaci (6BC) infection response’ (ID 11025) and four traits are associated with immune cell responses (ID 10201, 10466, 10238, 10236). In addition two traits described seizure responses (ID 10388, 10507), and one trait has not been disclosed yet (ID 13920). Within the early time chromosome 10 interval, three other traits exhibit their most significant QTLs: ‘3a,5a-THDOC in blood plasma 3
days after cycle 5 of chronic intermittent air vapor’ (ID13027) and two non-disclosed traits. The first trait may relate to stress responses in the central nervous system (ID 13292 and 13846).